Exp Mol Med.
2011 Apr;43(4):179-188. 10.3858/emm.2011.43.4.022.
Cathepsin L derived from skeletal muscle cells transfected with bFGF promotes endothelial cell migration
- Affiliations
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- 1Cardiovascular Research Institute, Yonsei University College of Medicine, Seoul 120-752, Korea. jhchung@yonsei.ac.kr
- 2Cardiovascular Product Evaluation Center, Yonsei University College of Medicine, Seoul 120-752, Korea.
- 3Yonsei Research Institute of Science for Aging, Yonsei University, Seoul 120-749, Korea.
- 4Severance Integrative Research Institute for Cerebral and Cardiovascular Diseases, Yonsei University Health System, Seoul 120-752, Korea.
- 5Institute of Health Science & Department of Food and Nutrition, College of Health Science, Korea University, Seoul 136-703, Korea.
- 6Severance Medical Research Institute, Yonsei University Health System, Seoul 120-752, Korea.
- KMID: 1104387
- DOI: http://doi.org/10.3858/emm.2011.43.4.022
Abstract
- Gene transfer of basic fibroblast growth factor (bFGF) has been shown to induce significant endothelial migration and angiogenesis in ischemic disease models. Here, we investigate what factors are secreted from skeletal muscle cells (SkMCs) transfected with bFGF gene and whether they participate in endothelial cell migration. We constructed replication-defective adenovirus vectors containing the human bFGF gene (Ad/bFGF) or a control LacZ gene (Ad/LacZ) and obtained conditioned media, bFGF-CM and LacZ-CM, from SkMCs infected by Ad/bFGF or Ad/LacZ, respectively. Cell migration significantly increased in HUVECs incubated with bFGF-CM compared to cells incubated with LacZ-CM. Interestingly, HUVEC migration in response to bFGF-CM was only partially blocked by the addition of bFGF-neutralizing antibody, suggesting that bFGF-CM contains other factors that stimulate endothelial cell migration. Several proteins, matrix metalloproteinase-1 (MMP-1), plasminogen activator inhibitor-1 (PAI-1), and cathepsin L, increased in bFGF-CM compared to LacZ-CM; based on 1-dimensional gel electrophoresis and mass spectrometry. Their increased mRNA and protein levels were confirmed by RT-PCR and immunoblot analysis. The recombinant human bFGF protein induced MMP-1, PAI-1, and cathepsin L expression in SkMCs. Endothelial cell migration was reduced in groups treated with bFGF-CM containing neutralizing antibodies against MMP-1 or PAI-1. In particular, HUVECs treated with bFGF-CM containing cell-impermeable cathepsin L inhibitor showed the most significant decrease in cell migration. Cathepsin L protein directly promotes endothelial cell migration through the JNK pathway. These results indicate that cathepsin L released from SkMCs transfected with the bFGF gene can promote endothelial cell migration.
Keyword
MeSH Terms
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Antibodies, Neutralizing/immunology
Cathepsin L/genetics/*metabolism
*Cell Movement
Cells, Cultured
Comet Assay
Dependovirus/genetics
Endothelial Cells/cytology/*metabolism
Fibroblast Growth Factor 2/genetics/immunology/*metabolism
Gene Transfer Techniques
Humans
Immunoblotting
JNK Mitogen-Activated Protein Kinases
Lac Operon/genetics
Mass Spectrometry
Matrix Metalloproteinase 1/biosynthesis/genetics
Muscle, Skeletal/*metabolism
Neovascularization, Physiologic
Plasminogen Activator Inhibitor 1/biosynthesis/genetics
RNA, Messenger/biosynthesis
Reverse Transcriptase Polymerase Chain Reaction
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